Fasting regulates mitochondrial function through lncRNA PRKCQ-AS1-mediated IGF2BPs in papillary thyroid carcinoma

Zhang, **; Zhong, Yong; Liu, Lin; Jia, Chengyou; Cai, Haidong; Yang, Jianshe; Wu, Bo; Lv, Zhongwei

doi:10.1038/s41419-023-06348-0

Fasting regulates mitochondrial function through lncRNA PRKCQ-AS1-mediated IGF2BPs in papillary thyroid carcinoma

Article
Open access
Published: 14 December 2023

Volume 14, article number 827, (2023)
Cite this article

Download PDF

You have full access to this open access article

Cell Death & Disease

Fasting regulates mitochondrial function through lncRNA PRKCQ-AS1-mediated IGF2BPs in papillary thyroid carcinoma

Download PDF

1575 Accesses
2 Citations
1 Altmetric
Explore all metrics

Abstract

Recurring evidence suggests that fasting has extensive antitumor effects in various cancers, including papillary thyroid carcinoma (PTC). However, the underlying mechanism of this relationship with PTC is unknown. In this study, we study the effect of fasting on glycolysis and mitochondrial function in PTC. We find that fasting impairs glycolysis and reduces mitochondrial dysfunction in vitro and in vivo and also fasting in vitro and fasting mimicking diets (FMD) in vivo significantly increase the expression of lncRNA-protein kinase C theta antisense RNA 1 (PRKCQ-AS1), during the inhibition of TPC cell glycolysis and mitochondrial function. Moreover, lncRNA PRKCQ-AS1 was significantly lower in PTC tissues and cells. In addition, PRKCQ-AS1 overexpression increased PTC cell glycolysis and mitochondrial function; PRKCQ-AS1 knockdown has the opposite effect. On further mechanistic analysis, we identified that PRKCQ-AS1 physically interacts with IGF2BPs and enhances protein arginine methyltransferases 7 (PRMT7) mRNA, which is the key player in regulating glycolysis and mitochondrial function in PTC. Hence, PRKCQ-AS1 inhibits tumor growth while regulating glycolysis and mitochondrial functions via IGF2BPs/PRMT7 signaling. These results indicate that lncRNA PRKCQ-AS1 is a key downstream target of fasting and is involved in PTC metabolic reprogramming. Further, the PRKCQ-AS1/IGF2BPs/PRMT7 axis is an ideal therapeutic target for PTC diagnosis and treatment.

FTO suppresses glycolysis and growth of papillary thyroid cancer via decreasing stability of APOE mRNA in an N6-methyladenosine-dependent manner

Article Open access 28 January 2022

LDHA induces EMT gene transcription and regulates autophagy to promote the metastasis and tumorigenesis of papillary thyroid carcinoma

Article Open access 01 April 2021

LncRNA GLTC targets LDHA for succinylation and enzymatic activity to promote progression and radioiodine resistance in papillary thyroid cancer

Article 08 April 2023

Introduction

Papillary thyroid carcinoma (PTC) is the most prevalent type of thyroid carcinoma, which is a common type of cancer with a high incidence rate among women [1, 2]. Prognosis for PTC has remained at ten years with the application of traditional surgical treatment options [3]. Consistent recurrence and metastasis necessitate the identification of new potential biological markers for PTC.

Calorie restriction (CR) has recently attracted a lot of attention due to its role in cancer treatment [4, 5]. Fasting mimicking diets (FMD) is a less aggressive type of CR [6,7,8], which have been considered as a treatment strategy in conjugation with chemotherapy [9]. During tumorigenesis, cells become more dependent on aerobic glycolysis rather than mitochondrial oxidative phosphorylation, allowing them to meet the energy and nutrient demands of actively proliferating cancer cells. Studies have indicated that FMD changes this dependency on glycolysis and decreases the proliferation of cancer cells, thus making FMD a potential treatment strategy for cancer [10, 11]. However, no study has been published to date on the underlying molecular effects of FMD on PTC.

Long non-coding RNA (lncRNA), a group of non-coding RNAs of approximately 200 bp length, interact with other RNAs or proteins and play important roles in transcriptional, post-transcriptional regulation and chromatin remodeling [12,13,14]. Previously, studies have identified dysregulation of lncRNA-protein kinase C theta antisense RNA 1 (PRKCQ-AS1) to be associated with cancer progression, including colorectal cancer, triple-negative breast cancer [15,16,17]. In the initial phase of the investigation, we accessed the Gene Expression Omnibus (GEO) database (GSE3678 and GSE3467) and observed that the expression of PRKCQ-AS1 in PTC is significantly diminished in the tissue adjacent to carcinoma. A recent study found that the PRKCQ-AS1 is highly upregulated in colorectal cancer patients [15], leading to an increase in proliferation and migration of CRC cells. However, there are no studies till date of lncRNA-PRKCQ-AS1’s role in PTC.

Insulin-like growth factor 2 binding proteins (IGF2BP 1, 2, and 3) are a family of evolutionarily conserved RNA-binding proteins known to be involved in many cellular processes such as proliferation, differentiation, migration, polarization, and metabolism [18]. Structurally, IGF2BP1, 2, and 3 appear similar to each other in the placement of the domains, with a molecular weight between 58 to 66 kDa [19]. With around 56% amino acid sequence similarity among the protein domains, they share at N-terminal two RNA-recognition motifs (RRMs) and four hnRNPK homology (KH) domains in the C-terminal. Studies based on in vitro flag tagged proteins have identified specifically IGF2BP1 binds to over 1000 mRNAs [20]. In many cancer types, IGF2BPs is identified to be highly expressed [21,22,23,24], however relatively less is known about its molecular and regulatory role in PTC.

Protein arginine methyltransferases (PRMTs) family of proteins catalyze the methyl transfer to arginine residues of protein substrates and have been linked to many types of cancer [25]. PRMT7 is one of PRMTs that could only catalyze formation of monomethyl arginine (Rme1) [26]. PRMT7 has been identified to play vital roles in breast and lung cancer by regulating metastasis and EMT transition [Full size image

PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction of PTC through targeting IGF2BPs in vitro

Assessment of IGF2BPs levels in PTC cell lines indicated that TPC-1 had the least level of IGF2BP3 while K1 had the highest IGF2BP2 and there is no difference in IGF2BP1 (Figs. 5A and S5C). So, we focused on IGF2BP2 and IGF2BP3 for subsequent experiments. Moreover, the transfection efficiency experiments confirmed that IGF2BP2/3 overexpression has higher IGF2BP2/3 expression levels, IGF2BP2/3 knockdown has lower expression levels (Fig. S6A). Cell population analysis indicated that overexpression of IGF2BP2 moved a huge population of cells to S phase, whereas overexpression of IGF2BP3 moved cells to G1 phase. Silencing of IGF2BP2/3 resulted in the opposite (Fig. S6B). Further to understand the dynamic interactions between PRKCQ-AS1 and IGF2BP2/3, we silenced PRKCQ-AS1 and overexpressed either IGF2BP2 or IGF2BP3 in PTC-1 and K1 cells. In IGF2BP2 overexpressing-PTC-1 cells, cell proliferation, colony formation unit, glucose uptake, lactate production, ECAR, glycolytic enzyme levels and ROS production increased, whereas OCR and mitochondrial ATP production significantly decreased, which was further augmented by PRKCQ-AS1 knockdown (Figs. 5B–I and S2C, left). Additionally, in IGF2BP3 overexpressing-K1 cells, all of these indicators were opposite, and reversed by PRKCQ-AS1 knockdown (Figs. 5B–I and S2C, right). Overexpression of IGF2BP2 significantly increased JC-1 mitochondrial monomers with fragmented mitochondrial structures and fission protein level in PTC-1 cells, while overexpression of IGF2BP3 increased mitochondrial aggregates with elongated mitochondrial structures and fusion protein level in K1 cells. However, this increase in aggregates, elongated structures, and fusion protein level were reversed by the silencing of PRKCQ-AS1 (Fig. 5J–L). All these results indicated that silencing of PRKCQ-AS1 enhanced the effects of IGF2BP2 overexpressing and reversed the effects of IGF2BP3 overexpressing confirmed that indeed PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction of PTC through targeting IGF2BPs.

PRKCQ-AS1 facilitates PRMT7 expression via interacting with IGF2BPs

To further understand the mechanism of regulating glycolysis and mitochondrial function, we assessed potential targets of IGF2BPs. Interestingly, we found a family of protein arginine methyltransferases (PRMTs) to be potential targets of IGF2BPs. It has been reported that IGFBP2 stabilizes PRMT6 mRNA [Full size image

PRMT7 is required for PRKCQ-AS1’s effect on regulation of PTC cell proliferation and cell cycle

Further, PRMT7 levels were assessed in the THCA database. Evidentially, PRMT7 levels were significantly lower in PTC patients, but there was no significant difference between stages of PTC. (Fig. 7A). Additionally, we assessed the mRNA expression level using qRT-PCR and confirmed significantly lower levels of expression in tumor samples (Fig. 7B). Assessment of PRMT7 levels in PTC cell lines indicated significantly low mRNA expression levels in all the PTC cells (Fig. 7C). Furthermore, the overexpression and knockdown efficiency of PRMT7 were confirmed both in mRNA and protein levels by qRT-PCR and western blot in K1 and TPC-1 cells (Fig. S7A). Silencing of PRMT7 reversed the effects of PRKCQ-AS1 overexpression on cell proliferation and colony forming of TPC-1 cells, and overexpression of PRMT7 reversed silencing of PRKCQ-AS1 similarly (Fig. 7D, E). Cell cycle analysis showed the effect of PRKCQ-AS1 overexpressed on cell population in G1 phase was reversed by PRMT7 silencing, additionally, similar effect occurs between PRKCQ-AS1 silencing and PRMT7 overexpressing (Fig. 7F). These results indicated that indeed PRMT7 is essential for PRKCQ-AS1’s effect on PTC.

PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction through IGF2BPs/PRMT7 signaling pathway in vitro

To understand further the effect of PRMT7 on glycolysis, we assessed glucose uptake, lactate production, ECAR, OCR, expression of glycolysis proteins, mitochondrial ATP, and ROS production in TPC-1 cells overexpressed or silenced for PRMT7 along with PRKCQ-AS1 (Figs. 8A–H and S2D). Additionally, we also assessed mitochondrial membrane potential, mitochondrial morphology, and expression of fusion and fission proteins in the above cells to understand the effect of PRMT7 on mitochondrial dysfunction (Fig. 8I–L). Results of these assays indicated the effect of overexpression of PRKCQ-AS1 that reduces glycolysis and mitochondrial dysfunction was reversed by silencing of PRMT7, and the effect of silencing of PRKCQ-AS1 that increases glycolysis and mitochondrial dysfunction was also reversed by overexpression of PRMT7. Furthermore, we assessed the effect of fasting on IGF2BP2/3 and PRMT7 mRNA and protein expression levels in TPC-1 and K1 cells under control and fasting medium. The results indicated that fasting decreased IGF2BP2 expression, which is exacerbated by PRKCQ-AS1 overexpression and alleviated by PRKCQ-AS1 knockdown; meanwhile, fasting promotes PRMT7 expression, while was promoted by PRKCQ-AS1 overexpression and inhibited by PRKCQ-AS1 knockdown in TPC-1 and K1 cells (Fig. S8A, B). On the contrary, fasting increases IGF2BP3 expression, and these are promoted by PRKCQ-AS1 overexpression and inhibited by PRKCQ-AS1 knockdown; and that, fasting promotes PRMT7 expression, while was promoted by PRKCQ-AS1 overexpression and inhibited by PRKCQ-AS1 knockdown in TPC-1 and K1 cells (Fig. S8C, D). In conclusion, these results clearly indicated that fasting regulates glycolysis and mitochondrial dysfunction via the PRKCQ-AS1/IGF2BPs/PRMT7 signaling pathway in PTC.

**Fig. 8: PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction through IGF2BPs/PRMT7 signaling pathway in PTC cells.**

Discussion

Cancer cells have immense energy needs for their increased proliferation and metastasis, which is typically met by increasing glycolysis and decreasing mitochondrial oxidative phosphorylation. This transformation in energy needs is termed as the Warburg effect [35, 36]. Relative literature reported that TPC1 and K1 carried mutations such as RET/PTC1 rearrangement or BRAF^V600E, and their effects in glycolysis are unknown in PTC [29, 30]. Numerous studies indicated that BRAF(V600E) mutant melanoma is one of the most glycolytic cancers. In BRAF(V600E) melanoma cells, activation of BRAF upregulates glycolysis and suppresses oxidative phosphorylation (OXPHOS) [37,38,39]. In papillary thyroid carcinoma, BARF^V600E is one of the most common genetic mutations associated with 40–80% PTC pathogenesis. And that, BRAF mutations activate the MAPK pathway, resulting in increases in cell proliferation, dedifferentiation, and apoptosis [40, 41]. Nevertheless, there are no reports on glycolysis through MARK pathway in PTC. Up to now, Gao et al. constructed BRAF^V600E-overexpressing and BRAF^V600E-knockdown thyroid cell lines for glycolysis experiments, the results indicated that the overexpression of BRAF^V600E enhanced aerobic glycolysis [29]. Couto et al. introduced that human thyroid cancer-derived cell lines [harboring RET/PTC or BRAF)] promoted STAT3 activation; STAT3 knockdown promoted aerobic glycolysis through increasing in glucose consumption, lactate production, and HIF1α [42]. However, the effect in details and the underlying mechanism of genes mutations on glycolysis in PTC has not been reported. In our study, TPC-1 cells were treated with SU11248, while K1 cells were treated with Vemurafenib before cultured in the fasting medium. The results indicated that BRAF^V600E and RET/PTC rearrangement has very little promotion on glycolysis (Fig. S1). Through the little promotion of gene mutations on glycolysis did not affect the inhibition of fasting on glycolysis in PTC, there are some discrepancies between these results and the reports. So, the effect of K1 cells harboring the BRAF^V600E mutation, or TPC-1 harboring the RET/PTC rearrangement on glycolysis needs to be further explored in depth, the molecular regulatory mechanisms need to be further investigated.

Fasting diets have been proven effective against many types of cancers [43, 44]. FMD has previously been identified to reduce this reliance on glycolysis, forcing cancer cells to reduce their proliferation and, as a result, the progression of tumorigenesis [11]. However, fasting in tumor progression and treatment has not yet been studied and reported in clinical study, including PTC, which is a clinical limitation of this manuscript. In this study, we observed a significant decrease in proteins involved in glycolysis under FMD. Additionally, we observed an increase mitochondrial fusion proteins and a decrease in fission proteins. Mitochondrial dynamics is critical for meeting cellular energy demands; mitochondrial fusion is frequently associated with increased ATP production, decreased ROS production, and impaired oxidative phosphorylation. Upregulation of fission proteins, on the other hand, is associated with increased ROS production and restricted oxidative phosphorylation [45]. Upregulation of the mitochondrial fission protein Drp1 has been linked to tumorigenesis in prostate cancer studies [46], whereas downregulation of fusion protein Mfn1 has been linked to tumorigenesis in hepatocellular carcinoma studies [47]. In our study, fasting increased fusion proteins such as Mfn1, Mfn2, and Opa1, as well as increased ATP production and decreased ROS production.

Rodent studies have shown that FMD reduces inflammation and cancer incidence while improving overall health and cognitive performance [48]. We wanted to further understand the molecular causes for FMD’s effect on PTC. We found a significant increase in lncRNA PRKCQ-AS1 after fasting, and low levels of lncRNA PRKCQ-AS1 have been linked to a poor prognosis and survival in PTC patients. Interestingly, there have only been two studies on lncRNA-PRKCQ-AS1 in colorectal cancer (CRC), with no studies on PTC. In CRC, both the studies indicated that high expression of PRKCQ-AS1 led to poor prognosis and survival among CRC patients [15, 17]. Our study indicated the opposite i.e., high expression led to increased survival among PTC patients. This variation in effects based on cancer type is not unexpected, as it has been observed with other non-coding RNA and cancer types. In studies on the lncRNA BANCR, knockdown of BANCR significantly reduced tumor growth in BALB/c mouse CRC models [49]. On the other hand, overexpression of BANCR reduced tumor growth in BALB/c PTC models [31]. Such differences between cancers suggest a potential need for additional functional studies in other models to better understand the role of the lncRNA. In both the in vitro and in vivo models used in this study, we observed a clear increase in lncRNA PRKCQ-AS1 under FMD led to a decrease in PTC cell proliferation and decrease in tumor size.

LncRNAs are now well accepted as molecules that interact with either RNA, DNA, chromatin, or proteins and thus functionally regulate their activity [50]. In this study, we observed that PRKCQ-AS1 binds strongly to IGF2BPs which we systematically illustrated to in turn regulate glycolysis and mitochondrial function in PTC. We used bioinformatics tools such as CatRAPID to further understand the molecular etiology of PRKCQ-AS1’s role in PTC and discovered that the lncRNA PRKCQ-AS1 strongly binds and regulates the expression levels of RNA-binding proteins IGF2BPs. IGF2BP1 and 3, which share 75% sequence homology, are significantly downregulated in the absence of PRKCQ-AS1, whereas IGF2BP2 is highly upregulated in the absence of PRKCQ-AS1. However, the role of IGF2BPs in cancer prognosis is equally controversial. In gastrointestinal cancer, for example, increased IGF2BP3 expression has been linked to a poor prognosis, increased cancer cell proliferation, and metastasis [51]. However, in ovarian cancer IGF2BP3 expression has been associated with increased survival [52]. In this study, we found that PRKCQ-AS1 binds to IGF2BP1 and 3 via the RNA-binding protein’s KH1 and 2 domains, whereas it binds to IGF2BP2 via the protein’s KH3 and 4 domains. Indeed, the contradictory regulation of lncRNA may be due to the fact that binding to the KH1 and 2 domains makes the RNA-protein complexes stronger and more stable than binding to the other domains [18].

Furthermore, we discovered that IGF2BPs do indeed regulate tumorigenesis by binding to and regulating PRMT7. Co-immunoprecipitation studies indicated that both IGF2BP2 and 3 binds to and regulates the expression of PRMT7. Importantly, we identified that increased levels of IGF2BP2 significantly correlated with decreased PRMT7 levels and increased tumorigenesis. However, increased IGF2BP3 levels significantly increased PRMT7 levels and decreased PTC tumorigenesis. However, relatively less is known about its role in cancer. Two studies on breast cancer and lung cancer had associated increased PRMT7 levels to poor prognosis and survival [27, 28]. However, till date no study has assessed its role and expression in PTC.

PRMT7 is a member of a protein family that is essential for the post-transcriptional modification of histones via arginine methylation [53]. As global epigenetic changes through modification of methylation patterns play key roles in cancer development and progression, PRMT7’s role is evident and clear in PTC [54]. Interestingly, when compared to normal thyroid tissue samples, proteins in PTC tissues are found to be globally hypomethylated [55]. More functional studies linking histone methylation, PTC, and PRMT7 are needed to confirm the epigenetic relationships among them. This is, however, the first study to contribute to the identification of fasting and lncRNA-PRKCQ-AS1, to assess the prognosis of PTC. Furthermore, we identified the role of PRKCQ-AS1/IGF2BPs/PRMT7 in PTC models with FMD, shedding light not only on the molecular functionality of FMD but also allowing identification of potential PTC treatment targets.

Materials and methods

Cell culture and treatment

Cells lines Nthy-ori3-1, BHP5-16, BCPAP, K1, and TPC-1 used in this study were purchased from the Shanghai Cell Bank (Type Culture Collection Committee, Chinese Academy of Sciences, Bei**g, China). Cells were cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin at 37 °C in a CO₂ incubator. The fasting mimic medium comprised of glucose-free DMEM supplemented with 0.5 g/L glucose and 1% FBS; fasting was mimicked by incubating cells in this medium for 24 h or 48 h. Before fasting medium, TPC-1 cells were treated with SU11248 (1 μM, 48 h); while K1 cells were treated with Vemurafenib, (1 μM, 48 h). Vemurafenib, a BRAF^V600E selective inhibitors, was purchased from Selleckchem (Houston, TX, USA). SU11248, a highly effective tyrosine inhibitor of the RET/PTC1 oncogenic kinase, was obtained from Pfizer, Inc.

Clinical samples

PTC tissue and adjacent thyroid tissues were collected from 107 patients from the Shanghai Tenth People’s Hospital affiliated with Shanghai Tongji University and approved by the Research Ethics Committee of the Shanghai Tenth People’s Hospital (Shanghai, China). Patient studies were conducted in accordance with the Declaration of Helsinki. Patients did not receive local or systemic treatment prior to surgery. Written informed consent for research purposes was provided by all patients. All samples were processed as needed, i.e., frozen at −80 °C for RNA extraction and samples were embedded and stored for immunohistochemical analysis. The use of these samples was approved by ethics committee of Shanghai Tenth People’s hospital. Written informed consent was obtained from all the patients involved in this study.

CCK 8 assay

Cells were initially seeded at 1500 cells/ well intensity onto a 96-well plate. After 24 h, CCK 8 assay was performed based on manufacturer’s instructions (CCK8; Do**do Laboratories, Japan). Briefly, the cells were incubated 10 µl CCK8 solution and incubated for 4 h in incubator and the absorbance was measured at 450 nm using a microplate reader.

Colony formation assay

Cells were seeded at a density of 1500 cells/well onto a 6-well plate. The cells were continued to be cultured till visible colonies were formed in the presence of complete growth medium. Cell colonies were finally fixed with methanol and stained with 0.1% crystal violet (Sigma, St. Louis, Mo) and counted.

RNA isolation and qRT-PCR

RNA isolation of the samples was performed using mRNeasy mini kit (Qiagen) based on the manufacturer’s instructions. Further, the RNA isolated were reverse transcribed into complementary DNA with M-MLV Reverse Transcriptase Kit (Promega, Madison, WI, USA). qRT-PCR was performed using SYBR green RT-PCR kit (ThermoFisher, Waltham, MA, USA) with the primers used in this study are provided in the Table S1.

Protein isolation and western blot

Cells and tissue samples were lysed using RIPA lysis buffer containing protease and phosphatase inhibitors. A total of 20 µg protein sample were loaded onto each well and resolved in a SDS gel. The samples were further transferred to a nitrocellulose membrane and the membrane were incubated with the below mentioned primary antibodies (IGF2BP1 (ab184305, Abcam); IGF2BP2 (ab124930, Abcam); IGF2BP3 (ab179807, Abcam); PRMT7 (ab181214, Abcam); MFN1 (ab129154, Abcam); MFN2 (ab205236, Abcam); OPA1 (ab157457, Abcam); DRP1 (ab184247, Abcam); FIS1 (ab156865, Abcam); MFF (ab129075, Abcam); PDK1 (ab202468, Abcam); LDHA (ab52488, Abcam); HK2 (ab227198, Abcam); GLUT1 (ab115730, Abcam); GPI (ab210753, Abcam); GAPDH (ab8245, Abcam); β-actin (ab8227, Abcam) after blocking with 5% skim milk. Finally, the membrane was incubated overnight with corresponding secondary antibodies before visualization using a chemiluminescence system (ECL).

OCR and ECAR

Oxygen consumptive rate (OCR) and extracellular acidification rate (ECAR) in TPC-1 and K1 cell lines were determined using a Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse Bioscience, USA). Briefly, 20,000 cells were seeded 96 plate/well and incubated overnight. Cells were then incubated for 1 h at 37 °C with glucose (100 mM), oligomycin (10 μM), and 2-deoxy-glucose (2DG, 1 M). ECAR and OCR levels were then measured to determine glycolytic capacity.

Glycolysis analysis

Glucose uptake analysis and lactate production analysis was performed using commercially available kits (Sigma-Aldrich, St. Louis, MO, USA), based on the manufacturer’s protocol. Briefly, 20,000 cells were seeded onto a 96 plate/well and incubated for 24 h before assessing the glucose uptake and lactate production.

Cell cycle analysis

Cell cycle analysis were performed using propidium iodide staining (PI, Signal way Antibody, #CA002) based on the manufacturer’s instructions. The cells were further analyzed using flow cytometry (Cell Lab Quanta, Beckman Coulter, USA).

Migration and invasion assays

The in vitro migration and invasion assays were performed using Transwell chambers. For the migration assay, 1 × 10⁵ TPC-1 or K1 cells with PRKCQ-AS1 overexpression were cultured in DMEM medium in the upper compartment of a Transwell chamber. For the invasion assay, 1 × 10⁵ TPC-1 or K1 cells with PRKCQ-AS1 silencing were resuspended in DMEM medium with 0.1% FBS in Matrigel-coated upper Transwell chambers. For both assays, the bottom chambers were filled with DMEM containing 10% FBS. The chambers were stained with 0.5% crystal violet. Migrated and invaded cells were counted under a microscope.

In vivo animal experiments

All the animal experiments performed in this study were in accordance with the procedures approved by the institutional review board of Shanghai Tenth’s People Hospital. In this study, we used BALB/c mice at the age 4–6 weeks obtained from Shanghai Tenth’s People Hospital. These mice were housed under standard pathogen-free environments with 12 h dark and light cycles. Mice were subcutaneously injected with TPC-1 or K1 cells into the right flank of the mice. When the tumors were palpable, the mice were randomly assigned to the control group and the FMD group. In the control group, the average daily consumption of mice was 14.9 kJ. The FMD consisted of three components, designated as the day 1 diet, day 2–3 diet, and day 4–7 diet, fed in this order. The day 1 diet contained only 50% of the calories of the normal intake. The day 2–3 diet contained only 10% of the calories of the normal intake. For the day 4–7 diet, the mice were fed their normal intake; this progression was followed by another cycle of the FMD. The animals had free access to water. The mice were further monitored and the subcutaneous tumors were measured throughout the 5 weeks. At the end, the tumors were dissected and the tumors were imaged and weighed. The samples were either fixed for immunohistochemical analysis or frozen for western or RNA analysis.

Immunohistochemistry

Immunohistochemical staining was performed on tumor tissue samples that are fixed in formalin and embedded in paraffin. After deparaffinization and rehydration, tissues were incubated with primary antibodies of Ki67 (Abcam) or IGF2BP1/2/3 (Abcam) at 4 °C overnight. Further, the tissues were treated with respective secondary antibodies. Finally, the sections were colored using diaminobenzidine before visualization under a light microscope (Leica DM IL, Germany).

Fasting plasma glucose and insulin measurement

At the end of the xenograft model experiment, tail blood samples were collected after 10 h of food restriction and used for the measurement of blood glucose and insulin concentrations were measured by using a handheld glucometer (Yuwell, Jiangsu, China) and a murine enzyme-linked immunosorbent assay kit (Shanghai Enzyme-linked Biotechnology Co., Shanghai, China).

Immunofluorescence staining

Cells were initially fixed using 4% paraformaldehyde for 15 minutes at room temperature. Further, after washing blocking of the samples were performed using 10% goat serum for 20 minutes at RT. Cells were then incubated with primary antibodies against IGF2BP1/2/3 overnight at 4 °C. Cells were thoroughly washed and incubated in respective secondary antibodies (Santa Cruz Biotechnology, USA) for 1 h at RT. Finally, the cells were stained with nuclear stain DAPI for 10 minutes before visualization using light microscope (Leica DM IL, Germany).

JC-1 staining

Cells were initially seeded onto a 96-well plate and incubated overnight. Cells were further treated with serum-free medium containing Cis with/without 2 mM NAC. The cells were further washed and incubated with 5,5′,6,′-tetrachloro-1,1′,3,3′ tetra- ethylbenzimidazolyl carbocyanine iodide (JC-1, Beyotime, China) dye for half hour. The cells were finally washed and visualized using light microscope (Leica DM IL, Germany).

RNA immunoprecipitation assay

TPC-1 or K1 cells were washed and lysed using RIP buffer at 4 °C for half hour. RIP assay was further performed based on manufacturer’s instructions (RIP lysis buffer, EMD Millipore, Billerica, MA, USA). The lysates were further treated with magnetic beads conjugated to the corresponding antibodies. The precipitated RNA was further isolated and quantified using qRT-PCR.

Co-immunoprecipitation

To detect protein–protein interactions, cells were lysed in 500 μl co-IP buffer supplemented with a cocktail of proteinase inhibitors. The lysates were centrifuged at 12,000 × g for 30 min, and the supernatant was used for immunoprecipitation with beads, which were preincubated with the corresponding antibodies. After incubation at 4 °C overnight, beads were washed three times with co-IP buffer. SDS sample buffer was added to the beads and the immunoprecipitates were used for western blot analysis.

Statistical analysis

All data shown in this study were analyzed using GraphPad Prism 8.0. Data are presented as mean ± standard error mean. Student T-test was used to assess statistical significance when comparing two groups. One-way analysis of variance was performed to assess significance when comparing multiple groups. P < 0.05 is considered as statistically significant.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.
Article PubMed Google Scholar
Hahn LD, Kunder CA, Chen MM, Orloff LA, Desser TS. Indolent thyroid cancer: knowns and unknowns. Cancers Head Neck. 2017;2:1.
Article PubMed PubMed Central Google Scholar
Cabanillas ME, McFadden DG, Durante C. Thyroid cancer. Lancet. 2016;388:2783–95.
Article CAS PubMed Google Scholar
Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science. 2009;325:201–4.
Article CAS PubMed PubMed Central Google Scholar
Mattison JA, Roth GS, Beasley TM, Tilmont EM, Handy AM, Herbert RL, et al. Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study. Nature. 2012;489:318–21.
Article CAS PubMed Google Scholar
Brandhorst S. Fasting and fasting-mimicking diets for chemotherapy augmentation. Geroscience. 2021;43:1201–16.
Article PubMed PubMed Central Google Scholar
Wei M, Brandhorst S, Shelehchi M, Mirzaei H, Cheng CW, Budniak J, et al. Fasting-mimicking diet and markers/risk factors for aging, diabetes, cancer, and cardiovascular disease. Sci Transl Med. 2017;9:eaai8700.
Article PubMed PubMed Central Google Scholar
Brandhorst S, Choi IY, Wei M, Cheng CW, Sedrakyan S, Navarrete G, et al. A periodic diet that mimics fasting promotes multi-system regeneration, enhanced cognitive performance, and healthspan. Cell Metab. 2015;22:86–99.
Article CAS PubMed PubMed Central Google Scholar
Campisi J, Andersen JK, Kapahi P, Melov S. Cellular senescence: a link between cancer and age-related degenerative disease? Semin Cancer Biol. 2011;21:354–9.
CAS PubMed PubMed Central Google Scholar
Liberti MV, Locasale JW. The warburg effect: how does it benefit cancer cells? Trends Biochem Sci. 2016;41:211–8.
Article CAS PubMed PubMed Central Google Scholar
Weng ML, Chen WK, Chen XY, Lu H, Sun ZR, Yu Q, et al. Fasting inhibits aerobic glycolysis and proliferation in colorectal cancer via the Fdft1-mediated AKT/mTOR/HIF1α pathway suppression. Nat Commun. 2020;11:1869.
Article CAS PubMed PubMed Central Google Scholar
Quinn JJ, Chang HY. Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet. 2016;17:47–62.
Article CAS PubMed Google Scholar
Harries LW. Long non-coding RNAs and human disease. Biochem Soc Trans. 2012;40:902–6.
Article CAS PubMed Google Scholar
Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell. 2009;136:629–41.
Article CAS PubMed Google Scholar
Cui G, Zhao H, Li L. Long noncoding RNA PRKCQ-AS1 promotes CRC cell proliferation and migration via modulating miR-1287-5p/YBX1 axis. J Cell Biochem. 2020;121:4166–75.
Article CAS PubMed Google Scholar
Zheng S, Fu W, Huang Q, Zhou J, Lu K, Gu J, et al. LncRNA PRKCQ-AS1 regulates paclitaxel resistance in triple-negative breast cancer cells through miR-361-5p/PIK3C3 mediated autophagy. Clin Exp Pharmacol Physiol. 2023;50:431–42.
Article CAS PubMed Google Scholar
Shademan M, Salanghuch AN, Zare K, Zahedi M, Foroughi MA, Rezayat KA, et al. Expression profile analysis of two antisense lncRNAs to improve prognosis prediction of colorectal adenocarcinoma. Cancer Cell Int. 2019;19:278.
Article PubMed PubMed Central Google Scholar
Bell JL, Wächter K, Mühleck B, Pazaitis N, Köhn M, Lederer M, et al. Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post-transcriptional drivers of cancer progression? Cell Mol Life Sci. 2013;70:2657–75.
Article CAS PubMed Google Scholar
Hansen TV, Hammer NA, Nielsen J, Madsen M, Dalbaeck C, Wewer UM, et al. Dwarfism and impaired gut development in insulin-like growth factor II mRNA-binding protein 1-deficient mice. Mol Cell Biol. 2004;24:4448–64.
Article CAS PubMed PubMed Central Google Scholar
Jonson L, Vikesaa J, Krogh A, Nielsen LK, Hansen T, Borup R, et al. Molecular composition of IMP1 ribonucleoprotein granules. Mol Cell Proteomics. 2007;6:798–811.
Article PubMed Google Scholar
Bellezza G, Cavaliere A, Sidoni A. IMP3 expression in non-small cell lung cancer. Hum Pathol. 2009;40:1205–6.
Article PubMed Google Scholar
Wang T, Fan L, Watanabe Y, McNeill PD, Moulton GG, Bangur C, et al. an RNA-binding protein as a potential therapeutic target for lung cancer. Br J Cancer. 2003;88:887–94.
Article CAS PubMed PubMed Central Google Scholar
Slosar M, Vohra P, Prasad M, Fischer A, Quinlan R, Khan A. Insulin-like growth factor mRNA binding protein 3 (IMP3) is differentially expressed in benign and malignant follicular patterned thyroid tumors. Endocr Pathol. 2009;20:149–57.
Article CAS PubMed Google Scholar
** L, Seys AR, Zhang S, Erickson-Johnson MR, Roth CW, Evers BR, et al. Diagnostic utility of IMP3 expression in thyroid neoplasms: a quantitative RT-PCR study. Diagn Mol Pathol. 2010;19:63–69.
Article CAS PubMed Google Scholar
Boffa LC, Karn J, Vidali G, Allfrey VG. Distribution of NG, NG,-dimethylarginine in nuclear protein fractions. Biochem Biophys Res Commun. 1977;74:969–76.
Article CAS PubMed Google Scholar
Zurita-Lopez CI, Sandberg T, Kelly R, Clarke SG. Human protein arginine methyltransferase 7 (PRMT7) is a type III enzyme forming ω-NG-monomethylated arginine residues. J Biol Chem. 2012;287:7859–70.
Article CAS PubMed PubMed Central Google Scholar
Cheng D, He Z, Zheng L, **e D, Dong S, Zhang P. PRMT7 contributes to the metastasis phenotype in human non-small-cell lung cancer cells possibly through the interaction with HSPA5 and EEF2. Onco Targets Ther. 2018;11:4869–76.
Article PubMed PubMed Central Google Scholar
Liu Y, Li L, Liu X, Wang Y, Liu L, Peng L, et al. Arginine methylation of SHANK2 by PRMT7 promotes human breast cancer metastasis through activating endosomal FAK signalling. Elife. 2020;9:e57617.
Article CAS PubMed PubMed Central Google Scholar
Gao Y, Yang F, Yang XA, Zhang L, Yu H, Cheng X, et al. Mitochondrial metabolism is inhibited by the HIF1alpha-MYC-PGC-1beta axis in BRAF V600E thyroid cancer. FEBS J. 2019;286:1420–36.
Article CAS PubMed Google Scholar
Kim DW, Jo YS, Jung HS, Chung HK, Song JH, Park KC, et al. An orally administered multitarget tyrosine kinase inhibitor, SU11248, is a novel potent inhibitor of thyroid oncogenic RET/papillary thyroid cancer kinases. J Clin Endocrinol Metab. 2006;91:4070–6.
Article CAS PubMed Google Scholar
Huang H, Weng H, Sun W, Qin X, Shi H, Wu H, et al. Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nat Cell Biol. 2018;20:285–95.
Article CAS PubMed PubMed Central Google Scholar
Cheng Y, Gao Z, Zhang T, Wang Y, **e X, Han G, et al. Decoding m(6)A RNA methylome identifies PRMT6-regulated lipid transport promoting AML stem cell maintenance. Cell Stem Cell. 2023;30:69–85.e67.
Article CAS PubMed Google Scholar
Li Z, Zhang J, Liu X, Li S, Wang Q, Di C, et al. The LINC01138 drives malignancies via activating arginine methyltransferase 5 in hepatocellular carcinoma. Nat Commun. 2018;9:1572.
Article PubMed PubMed Central Google Scholar
Shi Y, Niu Y, Yuan Y, Li K, Zhong C, Qiu Z, et al. PRMT3-mediated arginine methylation of IGF2BP1 promotes oxaliplatin resistance in liver cancer. Nat Commun. 2023;14:1932.
Article CAS PubMed PubMed Central Google Scholar
Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–33.
Article Google Scholar
DeBerardinis RJ, Chandel NS. We need to talk about the Warburg effect. Nat Metab. 2020;2:127–9.
Article PubMed Google Scholar
Shen S, Faouzi S, Souquere S, Roy S, Routier E, Libenciuc C, et al. Melanoma persister cells are tolerant to BRAF/MEK inhibitors via ACOX1-mediated fatty acid oxidation. Cell Rep. 2020;33:108421.
Article CAS PubMed Google Scholar
Parmenter TJ, Kleinschmidt M, Kinross KM, Bond ST, Li J, Kaadige MR, et al. Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis. Cancer Discov. 2014;4:423–33.
Article CAS PubMed PubMed Central Google Scholar
Haq R, Shoag J, Andreu-Perez P, Yokoyama S, Edelman H, Rowe GC, et al. Oncogenic BRAF regulates oxidative metabolism via PGC1α and MITF. Cancer Cell. 2013;23:302–15.
Article CAS PubMed PubMed Central Google Scholar
Scheffel RS, de Cristo AP, Romitti M, Vargas CVF, Ceolin L, Zanella AB, et al. The BRAF(V600E) mutation analysis and risk stratification in papillary thyroid carcinoma, Arch. Endocrinol Metab. 2021;64:751–7.
Google Scholar
Zhang P, Guan H, Yuan S, Cheng H, Zheng J, Zhang Z, et al. Targeting myeloid derived suppressor cells reverts immune suppression and sensitizes BRAF-mutant papillary thyroid cancer to MAPK inhibitors. Nat Commun. 2022;13:1588.
Article CAS PubMed PubMed Central Google Scholar
Couto JP, Daly L, Almeida A, Knauf JA, Fagin JA, Sobrinho-Simões M, et al. STAT3 negatively regulates thyroid tumorigenesis. Proc Natl Acad Sci USA. 2012;109:E2361–2370.
Article CAS PubMed PubMed Central Google Scholar
Caffa I, Spagnolo V, Vernieri C, Valdemarin F, Becherini P, Wei M, et al. A. Nencioni, Fasting-mimicking diet and hormone therapy induce breast cancer regression. Nature. 2020;583:620–4.
Article CAS PubMed PubMed Central Google Scholar
Nencioni A, Caffa I, Cortellino S, Longo VD. Fasting and cancer: molecular mechanisms and clinical application. Nat Rev Cancer. 2018;18:707–19.
Article CAS PubMed PubMed Central Google Scholar
Liesa M, Shirihai OS. Mitochondrial dynamics in the regulation of nutrient utilization and energy expenditure. Cell Metab. 2013;17:491–506.
Article CAS PubMed PubMed Central Google Scholar
** X, Wang J, Gao K, Zhang P, Yao L, Tang Y, et al. Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer. PLoS Genet. 2017;13:e1006748.
Article PubMed PubMed Central Google Scholar
Zhang Z, Li TE, Chen M, Xu D, Zhu Y, Hu BY, et al. MFN1-dependent alteration of mitochondrial dynamics drives hepatocellular carcinoma metastasis by glucose metabolic reprogramming. Br J Cancer. 2020;122:209–20.
Article CAS PubMed Google Scholar
Choi IY, Piccio L, Childress P, Bollman B, Ghosh A, Brandhorst S, et al. A diet mimicking fasting promotes regeneration and reduces autoimmunity and multiple sclerosis symptoms. Cell Rep. 2016;15:2136–46.
Article CAS PubMed PubMed Central Google Scholar
Ma S, Yang D, Liu Y, Wang Y, Lin T, Li Y, et al. LncRNA BANCR promotes tumorigenesis and enhances adriamycin resistance in colorectal cancer. Aging. 2018;10:2062–78.
Article CAS PubMed PubMed Central Google Scholar
Schmitt AM, Chang HY. Long noncoding RNAs in cancer pathways. Cancer Cell. 2016;29:452–63.
Article CAS PubMed PubMed Central Google Scholar
Jeng YM, Chang CC, Hu FC, Chou HY, Kao HL, Wang TH, et al. RNA-binding protein insulin-like growth factor II mRNA-binding protein 3 expression promotes tumor invasion and predicts early recurrence and poor prognosis in hepatocellular carcinoma. Hepatology. 2008;48:1118–27.
Article CAS PubMed Google Scholar
Noske A, Faggad A, Wirtz R, Darb-Esfahani S, Sehouli J, Sinn B, et al. IMP3 expression in human ovarian cancer is associated with improved survival. Int J Gynecol Pathol. 2009;28:203–10.
Article PubMed Google Scholar
Blanc RS, Richard S. Arginine methylation: the coming of age. Mol Cell. 2017;65:8–24.
Article CAS PubMed Google Scholar
Khan SA, Reddy D, Gupta S. Global histone post-translational modifications and cancer: Biomarkers for diagnosis, prognosis and treatment? World J Biol Chem. 2015;6:333–45.
Article PubMed PubMed Central Google Scholar
Ellis RJ, Wang Y, Stevenson HS, Boufraqech M, Patel D, Nilubol N, et al. Genome-wide methylation patterns in papillary thyroid cancer are distinct based on histological subtype and tumor genotype. J Clin Endocrinol Metab. 2014;99:E329–337.
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This study was sponsored by the National Natural Science Foundation of China (82002826, 81972543, 82071964) and Shanghai Shenkang Three-year Action Project (grant number: SHDC2020CR2054B), Shanghai Leading Talent Program sponsored by Shanghai Human Resources and Social Security Bureau (grant number: 03.05.19005), Shanghai Municipal Health Commission (grant number: GWV-10.1-XK09) and the Fasting mimics diet to promote T cell-mediated cytotoxicity in hepatocellular carcinoma project (2022JZ-15-Zhang **).

Author information

These authors contributed equally: ** Zhang, Yong Zhong.

Authors and Affiliations

Guangdong Provincial Key Laboratory of Tumor Interventional Diagnosis and Treatment, Zhuhai People’s Hospital, Zhuhai hospital Affiliated with **an University, **an University, 519000, Guangdong, China
** Zhang
Department of Nuclear Medicine, Shanghai Tenth People’s Hospital, Tongji University, 200072, Shanghai, China
** Zhang, Yong Zhong, Lin Liu, Chengyou Jia, Haidong Cai, Jianshe Yang & Zhongwei Lv
Center of Thyroid, Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 200233, Shanghai, China
Bo Wu

Authors

** Zhang
View author publications
You can also search for this author in PubMed Google Scholar
Yong Zhong
View author publications
You can also search for this author in PubMed Google Scholar
Lin Liu
View author publications
You can also search for this author in PubMed Google Scholar
Chengyou Jia
View author publications
You can also search for this author in PubMed Google Scholar
Haidong Cai
View author publications
You can also search for this author in PubMed Google Scholar
Jianshe Yang
View author publications
You can also search for this author in PubMed Google Scholar
Bo Wu
View author publications
You can also search for this author in PubMed Google Scholar
Zhongwei Lv
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

X.Z., Z.L., and B.W. performed study concept and design; Z.Y., L.L., C.J., and D.L. provided acquisition, analysis, and interpretation of data and statistical analysis; Z.Y. and J.Y. performed the experiments; Z.Y. and H.C. analyzed and interpretation of data, and statistical analysis; Z.Y. and J.Y. performed the statistical analysis and provided technical and material support; X.Z. and L.L. wrote and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to ** Zhang, Bo Wu or Zhongwei Lv.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the Shanghai Tenth People’s Hospital affiliated with Shanghai Tongji University. Informed consents were obtained from all individual participants included in the study. Animal experiments were approved by the Experimental Animal Care Commission of the Shanghai Tenth People’s Hospital affiliated with Shanghai Tongji University.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by Professor Stephen Tait

Supplementary information

Supplementary figure legends

Supplementary Table S1

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

Original western blots

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Zhang, X., Zhong, Y., Liu, L. et al. Fasting regulates mitochondrial function through lncRNA PRKCQ-AS1-mediated IGF2BPs in papillary thyroid carcinoma. Cell Death Dis 14, 827 (2023). https://doi.org/10.1038/s41419-023-06348-0

Download citation

Received: 01 December 2022
Revised: 01 November 2023
Accepted: 29 November 2023
Published: 14 December 2023
DOI: https://doi.org/10.1038/s41419-023-06348-0
Springer Nature Limited

Fasting regulates mitochondrial function through lncRNA PRKCQ-AS1-mediated IGF2BPs in papillary thyroid carcinoma

Abstract

Similar content being viewed by others

Introduction

PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction of PTC through targeting IGF2BPs in vitro

PRKCQ-AS1 facilitates PRMT7 expression via interacting with IGF2BPs

PRMT7 is required for PRKCQ-AS1’s effect on regulation of PTC cell proliferation and cell cycle

PRKCQ-AS1 regulates glycolysis and mitochondrial dysfunction through IGF2BPs/PRMT7 signaling pathway in vitro

Discussion

Materials and methods

Cell culture and treatment

Clinical samples

CCK 8 assay

Colony formation assay

RNA isolation and qRT-PCR

Protein isolation and western blot

OCR and ECAR

Glycolysis analysis

Cell cycle analysis

Migration and invasion assays

In vivo animal experiments

Immunohistochemistry

Fasting plasma glucose and insulin measurement

Immunofluorescence staining

JC-1 staining

RNA immunoprecipitation assay

Co-immunoprecipitation

Statistical analysis

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Ethical approval

Additional information

Supplementary information

Rights and permissions

About this article

Cite this article

Share this article

Search

Navigation